Both hemostatic plug formation and thrombosis require thrombin-dependent fibrin formation, platelet recruitment and aggregation. While hemostasis is an essential process, pathologic thrombosis can cause stroke, heart attack and other vasoocclusive diseases. Coagulation factors XI and XII (FXI, FXII) promote thrombin generation through the coagulation cascade. We found that both FXI and FXII contribute to experimental thrombosis, and epidemiologic data suggest that FXI deficiency is protective against deep vein thrombosis and ischemic stroke. Yet, unlike other coagulation factors, FXI-deficiency causes only a mild hemostasis disorder and FXII deficiency is apparently asymptomatic without a demonstrable role for FXII in normal hemostasis. Our original findings have opened a new window towards the development of new safe antithrombotic strategies. In this renewal application of my first R01 grant, we address the major unresolved questions on the role of FXI in thrombus formation, including activities that bypass the FIX- mediated intrinsic thrombin generation pathway. We will test our hypothesis that FXI also plays a key role in promoting thrombin generation through the inactivation of local endogenous anticoagulants. In Aim 1 we will characterize the mechanisms by which FXI-platelet crosstalk promotes the procoagulant activities of platelets during thrombus formation. We will test our hypothesis that FXIa promotes the clot formation through the inactivation of platelet tissue factor pathway inhibitor (TFPI). In Aim 2 we will define the role of FXIa in the prothrombotic activities of neutrophil extracellular traps (NETs), and will determine whether activation of the contact pathway by NETs promotes platelet activation and microaggregate formation distal to sites of thrombus formation under flow. In Aim 3 we will utilize in vitro and in vivo models to define the molecular mechanisms by which FXI promotes tissue factor-dependent coagulation on endothelial cells. The goal of the proposed studies is to better understand the role of contact activation in the initiation and propagation of thrombus formation, which may provide further rationale for therapeutic targeting of the FXI axis as a safer new strategy to combat thrombosis.